Investigations of the cellular mechanisms involved in glucose-induced insulin release have emphasized the importance of the endoplasmic reticulum in regulating intracellular Ca2+ concentrations. These studies have utilized a characterized endoplasmic reticulum-enriched fraction obtained from islet cells and the recent development of the digitonin permeabilized islet model which allows the measurement of CA2+ uptake and release by the endoplasmic reticulum, in situ. Recent studies have demonstrated the importance of myo-inositol 1,4,5-trisphosphate, a novel second messenger, and arachidonic acid in mobilizing Ca2+ from the islet-cell endoplasmic reticulum. This proposal is concerned with the identification of the cellular mechanisms involved in the regulation of Ca2+ uptake and efflux by the endoplamsic reticulum and its relation with glucose-induced insulin secretion. These studies will characterize further the role of inositol-trisphosphate; arachidonic acid, and its metabolites; sulfonylureas, and glucose and its glycoltic intermediates on the regulation of Ca2+ uptake and efflux by the islet endoplasmic reticulum. Mechanistically, we propose to evaluate the role of Ca2+ channels innvolved in Ca2+ efflux from the endoplasmic reticulum by using Ca2+ channel blockers and agonists; examining phosphorylation and dephosphorylation of a newly identified 60 kD phosphoprotein; and characterizing electrophysiologically the Ca2+ channel in endoplasmic reticulum vesicles incorporated into phospholipid bilayers. The correlation between intracellulr concentrations of arachidonic acid and insulin secretion will also be examined using gas chromatography-mass spectrometry. Models will be developed to demonstrate a direct effect of inositol-trisphosphate on insulin secretion. Finally, the characterization and identification of a phosphoprotein intermediate localized to the endoplasmic reticulum Ca2+-ATPase will be performed to correlate further the Ca2+-ATPase with Ca2+ transport by this organelle.